Prolonged Cell-cycle Arrest Associated with Altered cdc2 Kinase in Monocrotaline Pyrrole-treated Pulmonary Artery Endothelial Cells

Abstract

Monocrotaline pyrrole (MCTP), a metabolite of the pyrrolizidine alkaloid monocrotaline, is thought to ini- tiate damage to pulmonary endothelial cells resulting in delayed but progressive pulmonary interstitial edema, vascular wall remodeling, and increasing pulmonary hypertension. MCTP was previously shown to inhibit pulmonary endothelial cell proliferation and cause cell-cycle arrest in vitro . To determine the persistence of arrest and better characterize the cell-cycle stage at which it occurs, bovine pulmonary ar- tery endothelial cells (BPAEC) under differing growth conditions were exposed to low (5 m g/ml) or high (34.5 m g/ml) concentrations of MCTP for varying times. Flow cytometric cell-cycle analysis was coupled with Western blot and biochemical analysis of cdc2 kinase and measurements of cell size. MCTP treat- ment induced a G2 1 M phase arrest in 48-h exposed confluent BPAEC that persisted for at least 28 d and was associated with continued cellular enlargement. A short-duration MCTP exposure of confluent (low and high concentration) and log phase (high concentration) BPAEC caused persistent cell-cycle arrest for 1 wk, whereas a low-concentration exposure in log phase cells resulted in cell-cycle arrest with reversal 96 h after exposure. Western blot examination revealed that by 24 h of MCTP exposure, the phosphorylation state of cdc2 was consistent with the inactive form of the kinase (confirmed by biochemical assay); this al- teration persisted through at least 96 h of exposure. We conclude that MCTP induces a progressive irre- versible endothelial cell dysfunction leading to inactivation of cdc2 kinase and irreversible cell-cycle arrest at the G2 checkpoint. Thomas, H. C., M. W. Lamé, D. Morin, D. W. Wilson, and H. J. Segall. 1998. Prolonged cell-cycle arrest associated with altered cdc2 kinase in monocrotaline pyrrole-treated pul- monary artery endothelial cells. Am. J. Respir. Cell Mol. Biol. 19:129-142.